Project Summary Mitochondria, the site of a variety of important metabolic processes, are essential organelles of eukaryotic organisms. Pathological effects of reduced bioenergetic capacity and altered iron metabolism caused by mitochondrial dysfunction are common in human populations. Molecular chaperones play vital roles in the biogenesis of mitochondria. Two essential, highly conserved mitochondrial processes that depend upon the function of Hsp70 and J-protein molecular chaperones will be studied - translocation of proteins from the cytosol into the mitochondrial matrix and the generation of Fe/S clusters, critical co-factors for numerous enzymes. The goal of this proposal is to not only gain a better understanding of these two essential processes, but also to understand the mechanisms by which the action of Hsp70s and J-proteins are co-opted to make them competent to efficiently carry out specific roles in diverse biological processes. These two systems represent examples of the two basic features known to govern the ability of Hsp70s/J- proteins to function in diverse biological processes: localization to sites of action (protein import) and restricted client binding, rather than the promiscuous binding characteristic of function in protein folding (Fe/S cluster biogenesis). The vast majority of the hundreds of proteins of the mitochondrial matrix are synthesized on cytosolic ribosomes. Thus, efficient import of proteins is critical for mitochondrial function. The import motor required for driving proteins across the inner membrane into the matrix is composed of 5 essential components, with the matrix Hsp70, Ssc1, and its obligate J-protein co-chaperone, Pam18, at its core. Genetic, biochemical and structural approaches will be used, with a goal of understanding the regulated protein:protein interactions that have evolved to drive efficient translocation of proteins across the membrane. The results generated will also provide a framework for understanding Hsp70/J-protein machines in other systems, as mechanisms such as tethering regulated by interaction with client proteins are likely common regulatory strategies in chaperone systems. The mitochondrial matrix contains a set of essential proteins devoted to the biogenesis of Fe/S clusters. The clusters are assembled on the scaffold protein, Isu, via action of an assembly complex prior to transfer to recipient apo-proteins. The specialized J-protein:Hsp70 chaperone pair, Jac1:Ssq1, facilitates transfer of the cluster. Using biochemical interaction assays and exploiting mutant proteins having defects in their interactions with partner proteins, the mechanism by which binding of Jac1 initiates the transfer process will be studied to understand the kinetic switch between cluster formation and cluster transfer. Our work on this system will also serve as a model for a more general understanding of the means by which molecular chaperones modulate protein:protein interactions in other biolgical processes.